1. Field of the Invention:
This invention pertains to capillary electrophoretic resolution processes. In particular, capillary zone electrophoresis, such as that employed to separate proteins and DNA fragments, micellar electrokinetic capillary chromatography and related resolution processes, are enhanced by the application of one or more electric fields across the capillary tube, in addition to the linear field applied along the tube, by means exterior to the interior surface of the tube. The application of these potentials across the tube substantially prevents adsorption of macromolecules, such as proteins, and allows control over the electroosmotic flow rate, enhancing separation resolutions and efficiencies.
2. Background of the Prior Art:
Capillary Zone Electrophoresis (CZE) performs such functions as quality control of recombinant proteins, evaluation of the purity of synthetic peptides, studying serum proteins, evaluating DNA fragments, checking biological degradation, analyzing drugs, monitoring antibodies, and studying bioactive peptides with the resolving power of electrophoresis and the ease and speed of High Performance Liquid Chromatography (HPLC). The low volume capability, high separation efficiency, and sensitive detection schemes make CZE a powerful method for analytical biotechnology, a critical need for today's bioindustry.
A fundamental problem in CZE is controlling electroosmosis, the flow of solvent in an applied potential field. Under normal aqueous conditions with small binary electrolytes, the silica surface has an excess of anionic charge resulting from ionization of surface functional groups. The cationic counter-ions to these anions are in the diffuse layer adjacent to the capillary walls. The potential across the diffuse layer is termed the zeta potential. These hydrated cations migrate towards the cathode and drag solvent with them. Thus, the direction and rate of electroosmotic flow are dependent on the polarity and magnitude of the zeta potential at the capillary walls.
Electroosmotic flow affects the amount of time a solute resides in the capillary and in this sense both the separation efficiency and resolution are related to the direction and flow rate of electroosmosis. If the rate of electroosmotic flow is greater in magnitude and opposite in direction to the electrophoretic mobilities of all anions in the buffer, then all ions will migrate in the same direction. Thus, electroosmosis results in better resolution of anions which migrate against the electroosmotic flow. Conversely, cations will be more poorly resolved under these conditions. In fact, good resolution of substances having very similar mobilities can be achieved by balancing electroosmotic flow against electrophoretic migration. The invention of this application provides this control.
In addition to controlling electroosmosis, application of CZE to the separation of proteins is complicated by adsorption of the minute quantities of the protein sample onto the walls of the capillary. Such interactions result in band broadening and tailing, with greatly reduced separation efficiency. Reported attempts to eliminate this sorption involve deactivation of the silica capillaries by physically coating the capillary wall with methylcellulose, as well as via silane derivation. Because of the inherent difficulty of reproducibly deactivating the capillary surface, alternative methods employing dynamic reduction of protein/capillary interactions have been developed. These include the addition of chemical reagents to the separation buffer, as well as manipulation of the charges on the proteins and the silica capillary wall to prevent adsorption by Coulombic repulsion.
Similarly, capillary electrokinetics have been used to resolve non-ionic mixtures, as well as ionic species, through partition phenomena with micelles. The process involving the use of micelles is called the micellar electrokinetic capillary chromatography (MECC). See, e.g., Wallingford et al, Journal of Chromatoqraphy, 441, p. 299 et. seg. (1988).